Bis quaternary oximes



United States Patent 3,093,538 BIS QUATERNARY OXIMES Brennie E. Hackley, In, and Edward J. Poziomek, Edgewood, and George M. Steinberg, Baltimore, Md., assignors to the United States of America as represented by the Secretary of the Army .No Drawing. Original application Apr. 28, 1959, Ser. A

No. 809,578, new PatentNo. 3,077,476, datedMar. 12, 1963. Divided and this application Oct. 11, 1961, Ser.

3 Claims. (Cl. 167-65) (Granted under Title 35, us. coaermsz sec. 266) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon.

This is a division of'application Serial No. 809,578, filed April 28, 1959, now Patent No. 3,077,476, granted March- 12, 1963. x

This invention is directed to certain diquaternary pyridinium halide ox-imes which are useful as chemothera- -by anticholinesterases, particularly the=nerve gas known as GB or sarin, i.e., isopropyl methylphosphonofluoridate.

The invention relates to 1,1-polymethy1ene bis (4- tformylpyridinium) halide dioximes wherein the polya methylene group contains from 2 to 6 carbonatoms.

These compounds have the structural formula o I N\R OH NOH O H=N OH (I) where R is a polymethylene group having from two to six carbon atoms and X- 'is chloride, bromide or iodide.

This invention further includes compounds of Formula I, but in which R is a polyrnethylene group containing from seven to ten carbon'atoms. This invention further includes 1,1'(2,5 dimethyl-pphenylenedimethylene)- bis (4-formylpyridinium) halide dioximes of the formula ori N\ i CH-QNOHV 1 on=uon '7 i V (II) .peutic and prophylactic agents for mammals poisoned The invention also relates to 1,l'-(2-butenylene) bis (4-forrnylpyridinium) halide dioximes of the formula.

. GH=NOH (III) The invention further relates to 1,1'-poly-methylene bis .(3-fonmylpyridinium) halide dioximes of the formula (Iva) wherein R is a polymethy-lene group containing from 2 to 6 carbon atoms.- I

Another class of compounds included are the 1,1-(p- Patented June 11, 1963 ice phenylenedimethylene) bis (3-formylpyridinium) halide dioximes of the formula CH=NOH 0H=NOH (IVb) This invention further relates to unsymmetrical bisquaternary 4-formylpyridinium halide monoximes of the formula CH=NOH wherein R" represents either three lower alkyl groups or the hydrocarbon portion of the pyridine ring.

This invention also relates to the 1,l-trin1ethylene bis (3-amidooximopyridinium) halides While all these compounds are useful in varying degree for the purposes set out above, the different groups exhibit quite striking differences in effectiveness. In all the above formulas X- is chloride, bromide or iodide, which appear to be equivalents as to physiological action, except for their effect on solubility.

Wilson et al., in Patent No. 2,816,113, granted Dec.

,10, 1957, disclose a group of compounds which are effective as antidotes for treatment of mammals poisoned .by compounds of high anticholinesterase activity, such as the nerve gases diisopropylphosphorofiuoridate (DFP), isopropyl m'ethylphosphonofluoridate (GB), and O-eth-yl,

-N,N-dimethyl phosphoroamidocyanidate (GA), as well as other related organic phosphorus compounds, including many insecticides. The compound of the Wilson et al. group which has received by far the most attention is 2-formyl-1-methyl pyridinium iodide oxime, commonly known as 2-pyridine aldoxime methiodide or Z-PAM.

This compound is outstanding in its ability to reactivate,

in vitro, acetyl oholinesterase which has been inhibited by, for example, GB. Thus, even as compared to the very closely related 4-PAM, disclosed in Example II of the Wilson patent, 2-PAM shows much greater activity. With isopropyl methylphosphonylated acetylcholinesterase the rateconstant at pH 7.4 and 25 C. in the presence of 10- M acetylcholine is 2x10 per mole per minute for 2-PAM and 1.4 -10 per mole per minute forV4-PAM. The Wilson patent shows a high rate of survival in mice which had been poisoned with paraoxon and then treated with .2-PAM.

Nerve gas poisoning has been treated symptomatically with drugs which are pharamacologica-lly antagonistic to acetyl choline. Such a compound is atropine and it is at present the recommended remedy. Recently, Z-PAM has been reported to enhance considerably the activity of atropine in the chemotherapeusis of poisoning due .to organophosphorus compounds.

The compounds of Formula I above in which R contains from 2 to 6 carbon atoms are appreciably more effective than Z-PAM as reactivators of GB-inhibited acetylcholinesterase and also in enhancing the activity of atropine in both therapy and prophylaxis. When X- is bromide, the variation of the rate constant for the in vitro reactivation of GB inhibited eel acetylcholinesterase at pH 7.4 and 25 C. was found to be as follows:

TABLE 1 R Rate constant (1./moies/minntes) (CH2) 2 7X10 2): 6x10 (CH2); 6x10 (OHM 1x10 (CHzh 6X10 When administered in combination with atropine to animals poisoned with GB the order of eifectiveness was somewhat different. Under these conditions the compound in which R=(CH i.e., l,l-trimethylene bis (4-formylphyridinum) bromide dioxime also known as TMB-4, was most effective. In rats challenged with a 2 LD .dose of GB administered intravenously, all of a group of six animals survived the atropine-TMB-4 combination was administered intravenously immediately after poisoning. The atropine-Z-PAM combination saved only two of the group of animals. On the other hand, with dogs which were given a 20 LD dose of GB subcutaneously the survival ratios were the same (4/5) for the two treatments, which were given intravenously when symptoms appeared. However, the recovery time was much shorter for the surviving animals which received the TMB-4, i.e., 2 hours, as against 24 hours for those receiving the 2-PAM.

A summary of the reactivation rates and survival ratios for these compounds when administered therapeutically to rate together with atropine is as follows:

TABLE 2 R Reactivation Survival rate constant ratio (GB) 7X10 6/6 6X10 6/6 6x10 6/6 1X10 6/6 6X10 3/4 These compounds constitute our presently preferred group.

Our compounds may be employed prophylactically, i.e., injected before exposure to the anticholinesterase agent, e.g., GB, or therapeutically, i.e., injected subsequent to exposure.

The following seriesof experiments compares the effectiveness of our presently preferred compound, TMB-4, with Z-PAM applied to various animals by these two methods. The animals were poisoned by injections of GB as follows:

Mice 0.173 mg./kg. (LD Rats 0.126 mg./kg. (2 LD Rabbits:

Intravenous 0.340 mg./kg. (20 LD Subcutaneous 0.900 mg./k-g. (20 LD Dogs and cats:

Intravenous 0.440 mtg/kg. (20 LD Subcutaneous 0.900 rug/kg. (20 LD To minimize absorption effects both the GB and TMB-4 were ordinarily given intravenously. However, in the therapeutic tests on rabbits, dogs and cats, the GB was administered subcutaneously, since death from 20 LD intravenous dose of GB occurs so quickly that it is virtually impossible to give timely administration of the antidote.

Atropine, when administered, was included in the following amounts.

M'g/kg. Rats 4 Rabbits 2 Dogs and cats 0.5

The prophylactic doses were given within two minutes prior to the injection of the GB, the therapeutic doses so soon as poisoning symptoms were visible.

Table 3 shows the results.

TABLE 3 The recovery periods, i.e., time for disappearance of symptoms of poisoning, among survivors in the above tests, with atropine, were as follows.

TABLE 4 Z-PAM TMB-4 Animals Prophylactic Therapeutic Prophylactic Therapeutic Rats 60 min 15 min Rabbits 3 hr 30 min 2 hr. Cats 5 hr 5 hr 5 hr 24 hr Do s 24 hr 24 hr 1% hr 3 hr.

The compounds of Formula I in which R contains from 7 to 10 carbon atoms are less eifective than those of our preferred group. For these compounds the reactivation rate constant andthe survival ratio for rats (measured as given above) were as follows, X- being bromide.

TABLE 5 R Rate Survival Constant ratio (GB) 2x10 3 0/4 1.2 10 0/4 0 4 1.4X10 a 0/6 While these compounds were inelfective in vivo against GB, they were, together with 2-PAM, very eifective against certain other anticholinesterases, particularly that designated as VX by the US. Army Chemical Corps. All these compounds caused survival of all animals (surviva'l rates of 4/ 4 and 6/6) when administered therapeutically to rats challenged 'by 2 LD doses of VX.

The compounds of Formula II exhibited properties intermediate those of the two subgroups of Formula I.

When X'- was chloride the compound had the following properties. (In this and all following tables the survival ratios are those for rate challenged by 2 LD doses of G113 or (VX) and the oxime was employed therapeutica ly.)

Compounds of Formula III showed reactivation rates very close to those of our preferred group. Thus when X in Formula III is bromide the reactivation rate constant was 8x10 as compared to the value tor the member of our preferred group of 6x10 For the unsaturated member (Formula III) the survival ratio for rats challenged by GB was only 1/4 as compared to 6/ 6 for the saturated analogue (Formula I). Both gave complete survival (ratios of 4/4 and 6/6) for animals challenged by VX, however.

Compounds of Formula IV a showed anomalous properties.

They gave reactivation rates which were low, but survival ratios which were high as compared to Z-PAM, as shown by Table 6, X-- being bromide.

TABLE 6 R Reactivation Survival rate constant ratio (GB) (OHm 3. 6X10 4/4 (CH:|)5 4. 2X102 3/4 The compounds of group IVb, which are closely related to those of IVa, were somewhat less efiective. When X- wasbromide the compound had the following properties: Reactivation rate constant 2x10 survival ratio '(GB)-2/4.

The compounds or group V were another group in which the results of therapeutic treatment against GB were better as compared to Z-PAM then the reactivation rate constants would suggest, as shown by the following table, X- being bromide.

The compounds of Formulas VI, VII and VIII, while being of different structure are alike in exhibiting reactivation rate constants which are very low as compared to Z-PAM but giving high survival ratios as shown by Table 8, X" being bromide in each case.

TABLE 8 Formula No. Reactivation Survival rate ratio (GB) 69 4/4 Negligible 4/4 67 4/4 Preparation of Compounds '4-pyridinecarboxaldehyde oxime was prepared by warming on a steam bath a neutralized aqueous solution of 4-pyridinecarboxaldehyde and hydroxyla-miue hydrochloride. The oxime had a melting point of 130-1305 C. The 2- and 3-0'Ximes were produced by similar vmethods.

The quaternization to'produce dioximes was carried out by reacting the proper oxime with a 1, n =dihaloalkane, (0H X employing a 3:1 molar ratio 'of oxime to halide. The unsymmetrical quaternary monoximes were obtained by reacting the pyridine oxime with the appropriate omega-halopropyl quaternary salt in a 1.5 :1 molar ratio. Two procedures were utilized.

Procedure A: A mixture of the pyridine oxirne and halide was dissolved in sufiicient ethanol and refluxed for the period of time specified in Table 9.

Procedure B:' A mixture of the oxime and halide was dissolved in about 100 ml. of ethanol and heated in a 200 ml. capped pressure bottle (carbonated beverage type) for the length of time specified. The reaction mixtures were cooled to room temperature and the product removed by filtration. In several instances it was necessary to add absolute ether to effect complete precipitation. The products were recrystallized from ether. This procedure was usually employed because of its simplicity.

Table 9 gives the procedure, yields and melting points for representative compounds.

TABLE 9 For- Substituents Melting (m) mula Condi- Yield or decom- N o. tions percent position (d) Halide R R" point, C.

I Br (CH1), A, 31 hr 35. 0 300 111. I Br (CH5); B, 48 hr 88.2 238-241 (1 I Br (CHM B, 16 hr... 81.0 239-241 d I Br (0113) B, 95 hr 95.0 208-210 d I Br (CHzho B, 8 hr-.. 85.0 219-223 d II 01 B, 68 hr 70 300 n1 IVa Br (CHm B, 60 hr 68 208-211 m Iva... Br (CHm B, 60 hr 80 226-231 In IVb--. Br B, 20 83. 5 248-251 m V Br (C H )a B, 69 hr 43 230-231 d. V Br pygidine B, 64 10 223-226 d r n VIII... Br f B, 90 hr 16 201-203 d.

Further details regarding the preparation and properties of certain of our compounds are given in the following publications, by us and our associates:

Pyridine Aldoximes, by Edward I. Poziomek, Brennie E. 'I-Iackley, Jr., and George M. Steinberg, Journal of Organic Chemistry, vol. 23, pp. 714-717 (May 1958); and Chemotherapeutic Efiectiveness of Trimethylene bis (4-Formyl Pyridinium Bromide) Dioxime in Anticholinesterase Poisoning, by Edmund Bay, S. Kropp, and L. 'F. Yates, Proceedings of the Society for Experimental Biology and Medicine, vol. 98, pages 107- 109 (May 1958). These articles are to be considered incorporated by reference in this specification.

While we have shown a number of specific examples of compounds and their use, it will be obvious that various changes can be made without departing from our invention, which is defined by the following claims.

We claim:

1. A method of therapeutically treating a mammal which has been poisoned by a compound having high anticholinesterase activity which comprises injecting a composition consisting of atropine and an unsymmetrical bis quaternary 4-for-myl pyridinium halide monoxime of the formula CH=NOH wherein R is selected from the class consisting of three lower alkyl groupsand the hydrocarbon portion of the 3. A method of therapeutically treating a mammal pyridine ring. which has been poisoned by a compound having high 2. A method of therapeutically treating .a mammal anticholinesterase activity which comprises injecting a which has been poisoned by a compound having high composition consisting of atropine and an unsymmetrical anticholinesterase activity which comprises injecting a 5 bis quaternary 3-formyl pyridinium halide monoxime composition consisting of atropine and an unsymmetrical of the formula bis quaternary 2-formyl pyridinium monox-ime halide of References Cited in the file of this patent wherein X- is selected from the class consisting of chlo- Poziomek, Am. Chem. Soc. Abst. of Papers, 132nd ride, bromide and iodide. meeting, 1957, page 16-0. 

1. A METHOD OF THERAPEUTICALLY TREATING A MAMMAL WHICH HAS BEEN POISONED BY A COMPOUND HAVING HIGH ANTICHLINESTERASE ACTIVITY WHICH COMPRISES INJECTING A COMPOSITION CONSISTING OF ATROPINE AND AN UNSYMMETRICAL BIS QUANTERNARY 4-FORMYL PYRIDINIUM HALIDE MONOXIME OF THE FORMULA 